Description of the male reproductive sys

Abstract

The male gonopores, male reproductive apparatus, spermatophore and spermatozoa of the Mediterranean hermit crab Paguristes eremita are described, using interference phase microscopy, scanning electron microscopy and transmission electron microscopy. A correlation is made between the gonopore morphology and the different kinds of setae accompanying them, and the reproductive biology of these crabs. Each testes merges into a tubular duct made up of four zones: (1) the collecting tubule with free spermatozoa; (2) the proximal zone, where the ampulla of the spermatophores starts to be formed; (3) the medial zone, where the ampulla is completed, the stalk lengthens and the pedestal is formed; (4) the distal zone, where the mature spermatophores are stored. The sizes of the different parts of the spermatophore and of the sperm are given and their exterior morphology and ultrastructure described and compared to congeners. The morphology of the gonopore, male reproductive system, spermatophore and spermatozoa of P. eremita are species-specific, clearly distinguishing the species from the other members of the family. The available spermatozoal and spermatophore data is used to place P. eremita within a sperm phylogeny of the hermit crab family Diogenidae. & 2010 Elsevier GmbH. All rights reserved.

Keywords: Hermit crab; Reproductive tract; Morphology; Ultrastructure; Phylogenetic analysis

1. Introduction

mechanisms, and the structure of spermatozoa and spermatophores have been studied extensively in the

Recently, comparisons of the functional morphology Decapoda and provide useful information on phyloge- of genitalia and subsequent sperm transfer and storage

netic relationships and evolutionary divergence (see Bauer 1986, 1991 ; Kronenberger et al. 2004 ). In particular, the reproductive apparatus of hermit crabs,

fax: þ39 011 6704508.

made up of gonopores (sometimes sexual tubes), testes

E-mail address: santina.tirelli@unito.it (T. Tirelli).

and vasa deferentia containing spermatophores and

0044-5231/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jcz.2010.01.001

T. Tirelli et al. / Zoologischer Anzeiger 248 (2010) 299–312

spermatozoa, show a species-specific morphology, that Caorle, Veneto, Mediterranean Sea (45134’58 00 N, has been successfully used in recent phylogenetic studies

E) during summer 1998. After collection, the ( Tudge 1997 ; Tirelli et al. 2008 ).

specimens were immediately preserved in 4% formalin The hermit crab family Diogenidae is comprised of 20

in seawater or in 2.5% glutaraldehyde. genera and approximately 1200 species ( McLaughlin,

For each individual the cephalothorax length (CL) 2003 ; Lemaitre. pers. comm.) and for 13 genera

was measured from the tip of the rostrum to the ( Allodardanus Haig & Provenzano, 1965, Aniculus

V-shaped groove at the posterior edge of the cepha- Dana, 1852, Bathynarius Forest, 1989, Cancellus

lothorax, with a stereomicroscope fitted with a grad-

H. Milne Edwards, 1836, Ciliopagurus Forest, 1995,

uated eyepiece.

Isocheles Stimpson, 1858, Paguropsis Henderson, 1888, The use of animals was approved by the appropriate Petrochirus

animal care review committee at the Dipartimento di McLaughlin, 2002, Pseudopagurus Forest, 1952, Stra-

Biologia Animale e dell’Uomo dell’Universita` di Torino tiotes Thomson, 1899, Tisea Morgan & Forest, 1991 and

(Italy), where the study was carried out. Trizopagurus Forest, 1952) the reproductive apparatus morphology is still unknown or incompletely known ( Mantelatto et al. 2009 ) and could be very important for

2.2. Microscopy and morphometry comparative studies.

Observations were made under interference phase- Paguristes eremita (Linnaeus, 1767) is a common contrast (IPM) light microscopy, scanning electron Mediterranean hermit crab belonging to the family microscopy (SEM), and transmission electron micro- Diogenidae. It lives on sandy bottoms of the infralittoral scopy (TEM). Five specimens were prepared for light zone. Its larval distribution ( Thiriot 1974 ), shell use, and microscopy observations, three specimens for SEM epibiotic relationships ( Tirelli et al. 2006a ) are well observations, and two specimens for TEM observations known, but no complete description of the male according to standard protocols described in Tirelli et al. reproductive apparatus is available for this species.

(2006b, 2007) .

Therefore the aim of this paper is to expand the The distal part of the vas deferens was used for the knowledge of the reproductive morphology of P. eremita spermatophore analysis. A regression analysis was by analyzing and describing the male gonopores, the performed to compare the width of the last portion of reproductive tract, the mature spermatophores and their the distal part of the vas deferens (VDW) and the size stages of maturation, studying the sperm ultrastructure,

(CL) of the crabs.

and comparing them with similar structures in other Twenty spermatophores from each of 5 specimens used members of the family Diogenidae. A second phyloge- for IPM analysis were measured. The following measure- netic analysis of selected Diogenidae is performed, ments were taken: ampulla length; ampulla width; stalk adding to a previous one ( Tirelli et al. 2008 ) characters length; stalk width; pedestal length; pedestal width. A concerning P. eremita sperm and spermatophore ultra- regression analysis was performed to compare the ampulla structure. dimensions and the width of the distal vas deferens. This paper is part of a series that aims to describe the Measurement of the different layers making up the male reproductive apparatus of four of the most spermatophore wall were performed. common Mediterranean hermit crab species: Calcinus Lastly, the different regions of the spermatozoa for tubularis Linneus, 1967 (see Tirelli et al. 2006b ), each one of the 2 specimens observed under TEM were Clibanarius erythropus Latreille, 1818 (see Tirelli et al.

measured.

2007 ), Diogenes pugilator Roux, 1829 (see Tirelli et al. 2008 ) and Paguristes eremita (present study). In particular, for P. eremita we present new research that,

2.3. Phylogenetic analysis

together with the limited data already published ( Mouchet 1931 ), contributes to the knowledge of the

The diogenid taxa included in this analysis are taken reproductive apparatus of this species.

from Tudge (1997) , except for the following four hermit crabs (listed with their source): Calcinus tubularis (from Tirelli et al. 2006b ), Calcinus tibicen Herbst, 1791 (from Amadio and Mantelatto 2009 ), Clibanarius vittatus

2. Materials and methods

Bosc, 1802 (from Matos et al. 1993 ; Hess and Bauer 2002 ), Diogenes pugilator (from Manjo´n-Cabeza and

2.1. Specimens Garcia Raso 2000 ; present study), and Loxopagurus loxochelis Moreira, 1901 (from Scelzo et al. 2004, 2006 ).

Ten adult specimens (indicated by 410.00 mm The current analysis uses 19 of the 32 characters listed carapace length and gonopores present) of P. eremita

by Tudge (1997) : character numbers 3–10, 12, 14–18, 21, were collected on a sandy bottom at 9 m depth from

29, 31, and 32, plus a new one regarding presence or

T. Tirelli et al. / Zoologischer Anzeiger 248 (2010) 299–312

absence of the peripheral acrosome zone (0=absent,

3. Results

1=present). The outgroup taxon, Pagurus bernhardus Linneus

3.1. Gonopores

1758 (family Paguridae) is chosen because of its sister- group relationship to the diogenid taxa (plus the

The gonopores of P. eremita are paired structures, on coenobitids) in the phylogeny of Tudge (1997) .

both the left and right coxa of P5, and are flush with The matrix is available, upon request, from the first

each coxal surface. Each gonopore is a circular pore author (TT).

covered by a membranous operculum, possibly hinged The parsimony analysis was performed using PAUP

mesially. A ventral view of a single coxa with gonopore version 4.0b10 ( Swofford 2002 ) on a data matrix

A. At a short distance from the established as a Nexus file by MacClade 4.08 ( Maddison

is shown in Fig. 1

gonopore there are two kinds of setae: annulate without and Maddison 2005 ). Heuristic searches found the most

setules and annulate with setules. In particular the setae parsimonious trees, with the following options: the

are assembled into four isolated groups of three setae starting trees were obtained by stepwise addition

each. The three setae making up each group ( Fig. 1 B) (random addition sequence, RAS, 100 replicates) and

differ from each other: one is short, annulate without then rearranged by the branch swapping algorithm tree-

stw=5.0070.20 mm; bisection-reconnection (TBR).

setules

(stl=45.0470.37 mm,

mean71 SD, n=6) ( Fig. 1 C), one is medium, annulate All characters were treated as unordered and initially

with a setule in the distal third (stl=100.0871.25 mm, equally weighted, but then reweighted according to their

D) and own rescaled consistency index (RC), using the iterative

stw=9.0070.24 mm; mean71SD, n=6) ( Fig. 1

one is long, annulate with setules along the apical protocol of Farris (1969, 1988) .

portion (stl=210.0871.69 mm, stw=9.8870.60 mm; Support for tree topology was evaluated by perform-

E and F). The longest setae ing a bootstrap analysis (500 replicates, 10 RAS, TBR).

mean71SD, n=6) ( Fig. 1

are annulate without setules (stl=300.5472.45 mm,

21 21 m

54 54 m

Fig. 1. Paguristes eremita: SEM of male gonopore morphology. (A) Right coxa of pereopod 5 showing gonopore closed by the membranous operculum and very long setae along the coxal edge (arrow); (B) group of isolated setae composed of 3 setae; (C) short seta showing the basal grooves, the annulus and the flattened distal portion; (D) medium seta showing the annulus and the distal portion with the setule (arrow); (E) basal portion of long setae showing the grooves; (F) detailed view of the setules (arrow) on a long seta.

T. Tirelli et al. / Zoologischer Anzeiger 248 (2010) 299–312

stw=9.9670.24 mm; mean71SD, n=6) and constitute epithelium is generally composed of cells containing

a thick row, along the coxal edge ( Fig. 1 A). cytoplasmic organelles, such as cisternae of the rough All four types of setae have a central axis, which is

endoplasmic reticulum filled with secretory products slightly curved and flattened (almost spatula shaped) in

C) occupying a large fraction of the cell, some the distal portion, and a distinct annulus dividing them

( Fig. 2

Golgi bodies, and many small vesicles. The secretory into a basal portion showing slight longitudinal grooves

material produced by the epithelium is evident in the and a distal smooth portion ( Fig. 1

vasa deferentia lumen ( Fig. 2 D). The vas deferens is composed of four zones: (1) highly coiled narrow collecting tubule, (2) proximal, (3) medial,

3.2. Testes, vasa deferentia and stages of and (4) distal zones ( Fig. 3 A). The proximal and the medial spermatophore maturation

zones are characterized by a very high number of coils. The amorphous sperm mass flows embedded in the The male reproductive tract is composed of paired

seminal fluid from the testes to the adjacent collecting gonads, consisting of testes and vasa deferentia, located

tubule ( Fig. 3 B). In the proximal zone, the sperm mass is dorsally in the pleon. The testes lie dorsal to the gut, on

subdivided by the dense primary spermatophore layer, the large hepatopancreas, while the vasa deferentia

which envelops it. In between the proximal and the descend vertically into the hepatopancreas and release

medial zones, the aggregated spermatozoa are separated the spermatophores externally through the paired

and compartmentalized to form the ampullae ( Fig. 3 C). gonopores.

In the medial zone, the stalk and foot are built and the The paired, lobe-like testes are composed of cystic

stalk lengthens ( Fig. 3 D). The mature spermatophores structures. Each testis merges into a tubular duct.

E and F) then are stored in the distal zone. Throughout their length the vasa deferentia are com-

( Fig. 3

CL was 11.2071.16 mm and VDW was 292.0070.05. posed of two main layers: an inner secretory epithelium

VDW was positively correlated with the size of the crab ( Fig. 2

A) and an outer muscular layer ( Fig. 2 B). The

( r 2 =0.437; F=11.658; po0.05).

Fig. 2. Paguristes eremita: TEM of spermatophore and reproductive tract. (A) Detail of the reproductive tract ultrastructure showing a cell of the secretory epithelium (se) bounding the lumen (l); (B) detail showing the muscular layer (ml) external to the secretory epithelium (se); (C) epithelial cell with highly developed rough endoplasmic reticulim (rer); (D) lumen filled with the secretory material (sm) produced by the epithelial cells; (E) spermatophore wall ultrastructure showing the granular layer (gl) the fibrillar layer (fl) and the electron-dense patches (edp).

T. Tirelli et al. / Zoologischer Anzeiger 248 (2010) 299–312

Fig. 3. Paguristes eremita: stereomicroscopy and interference phase-contrast microscopy of male reproductive apparatus and stages of spermatophore maturation. (A) Testes (t), coiled collecting tubule (c), proximal (p), medial (m), and distal (d) zones; (B) amorphous sperm (sp) mass in the collecting tubule; (C) agglutinated spermatozoa shaped to form the ampullae (a); (D) distal zone with each spermatophore composed of an ampulla (a) with spermatozoa (sp), stalk (s) and foot (f), bound to each other by the basal cord (bc); (E) detail of a mature spermatophore showing the shape of the ampulla (a) with spermatozoa (sp) and the stalk (s); (F) spermatophore ampulla (a) with spermatozoa (sp) inside.

3.3. Spermatophore ampulla, we performed a regression analysis to compare the ampulla length (AL) and the distal vas deferens

The spermatophore of P. eremita ( Fig. 3 width (VDW). No correlation between AL and VDW sperm-filled ampulla elevated on a stalk, which is

was found.

connected to a pedestal or foot. The ampulla is generally An accessory ampulla, associated with the main cylindrical in shape but its basal end (the one towards

ampulla, is present only in spermatophores, which have the stalk) is narrower ( Fig. 3 E), therefore the whole

not yet ended the maturation process, therefore have not ampulla has an upside down pear shape. The ampulla is

yet fully developed the stalk.

45.277.3 mm ( n=100) wide and 92.677.2 mm (n=100) Observations of the spermatophore wall ultrastruc- long. The stalk is 49.276.5 mm ( n=6) in length, and

E) show an inner, slightly granular and 6.571.4 mm ( n=6) in width, and the width is constant

ture ( Fig. 2

moderately electron-dense layer, showing in its middle for most of the stalk length. The stalk gradually thickens

some quite electron-dense patches, followed by a thin in its distal portion to envelop the ampulla. The pedestal

outer fibrillar layer. The fibrillar layer is just 1/10 as has a characteristic shape: it resembles a half-moon with

thick as the granular one, being 0.12970.002 mm ( n=2) the convexity towards the stalk ( Fig. 3 D). It is

while the granular one is 1.01270.107 mm ( n=2). 2.570.0 mm ( n=6) long and 10.871.3 mm (n=6) wide. The stalk inserts at around half of the pedestal width. The mature spermatophores in the reproductive duct are

3.4. Spermatozoa

enveloped by protective layers and they are connected by a basal cord ( Fig. 3 D).

A) are composed of an almost As the only spermatophore portion measured in the

Spermatozoa ( Fig. 4

conical acrosomal vesicle capped by an operculum. At spermatophores coming from all the specimens was the

the base of the acrosomal vesicle, three arms are

T. Tirelli et al. / Zoologischer Anzeiger 248 (2010) 299–312

Fig. 4. Paguristes eremita: TEM of spermatozoa. (A) acrosomal vesicle of spermatozoon capped by the operculum (op), subopercular zone (sub), perforatorial chamber (per) surrounded by the inner acrosome zone (inn), the outer acrosome zone (out) and the peripheral acrosome zone (pa); (B) perforatorial chamber, showing dense tubules (dt), surrounded by the inner (inn), outer (out) and peripheral (pa) acrosome zones; (C) detail of the perforatoral chamber clearly showing the dense tubules (dt); (D) basal portion of the spermatozoa showing the connection between the cytoplasm and the perforatorial chamber (per), the outer (out) and peripheral (pa) acrosome zones and the cytoplasmic region with degenerating mitochondria (mit) and base of the microtubular arms (ar); (E) detail of the basal portion of the sperm with mitochondria (mit) and base of the microtubular arms (ar) and acrosome with peripheral (pa) and outer (out) acrosome zones.

Table 1. Various dimensions of the spermatozoa of Paguristes eremita. Maximum width (mm)

Length (mm) pa

acr Total n

3.360 4.395 4.744 s.d.

0.285 0.139 0.136 Abbreviations: acr=acrosome; cyt=cytoplasm; pce=perforatorial chamber entrance; inn=inner acrosome zone; n=number of spermatozoa

measured; out=outer acrosome zone; pa=peripheral acrosome zone; per=perforatorial chamber; s.d.=standard deviation.

positioned to form a 1201 angle between each other. and then tapers anteriorly. Inside the chamber dense P. eremita total sperm length is 4.7 mm, total sperm

B and C), with a tendency to be arranged width is 3.6 mm, acrosomal length is 4.4 mm, and

tubules ( Fig. 4

longitudinally, occur in the middle portion. The outer acrosomal width is 3.5 mm (more detailed sperm cell

A and B) is less electron-dense than dimensions are shown in Table 1 ).

acrosome zone ( Fig. 4

the chamber wall and slightly more granular in appear- The acrosomal vesicle is posteriorly penetrated by the

ance. In the basal portion of the perforatorial chamber, perforatorial chamber ( Fig. 4 A–D), which in longitudinal

the outer acrosome zone is close to the chamber itself, section is conical in shape. This is an invaginated column,

then, from approximately half the sperm length, it constricted at its base, which swells to form a kind of bulb

extends anteriorly separated from the perforatorial

T. Tirelli et al. / Zoologischer Anzeiger 248 (2010) 299–312

chamber by the inner acrosome zone ( Fig. 4 A). This last The male gonopores are closed by a thin, possibly zone is slightly granular and more electron-dense than the

non- or weakly calcified membrane termed an opercu- outer acrosome zone. Exterior to the outer acrosome

lum. Although indistinct, a hinge line is possibly visible zone there is the peripheral acrosome zone ( Fig. 4 A),

on the mesial side of the membranous operculum. extending around the periphery of the acrosome vescicle,

A similar hinge has been reported in male hermit crabs from the operculum to the base of the perforatorial

C. vittatus (see Hess and Bauer 2002 ), Diogenes chamber. This is a homogeneous, finely granular and

of

pugilator (see Manjo´n-Cabeza and Garcia Raso 2000 ) moderately electron-dense zone, barely distinguishable in

C. erythropus (see Tirelli et al. 2007 ) and in some the basal portion of the spermatozoa. The peripheral

and

carideans of the genera Heptacarpus Holmes, 1900 (see acrosome zone progressively widens anteriorly, filling the

Bauer 1976 ), Procaris Chace & Manning, 1972, Lysmata gap left by the outer acrosome zone, which, in turn, gets

Risso, 1816 (see Felgenhauer et al. 1988 ), and Chorismus narrower. The operculum is a highly electron-dense cap

Bate, 1888 (see Mascetti et al. 1997 ). Probably, as that anteriorly delimits the acrosomal vesicle ( Fig. 4 A);

C. erythropus ( Tirelli et al. 2007 ), the the area beneath the operculum makes up a granular

already noted for

operculum is used as protection from dehydration moderately electron-dense subopercular zone ( Fig. 4 A).

during emersion periods. When copulation occurs, It is of similar appearance to the inner acrosome zone but

spermatophores are extruded due to the internal is slightly more electron-dense. The cytoplasm envelops

pressure exerted on the operculum, which then opens the base of the acrosomal vesicle and is directly connected

outwards with a door-like mechanism. Nevertheless, as with the perforatorial chamber contents ( Fig. 4

no direct observations have ever been made on In the cytoplasm there are spherical mitochondria, both

A and D).

opercular opening, chemical dissolution, muscular cristate and degenerate ( Figs. 4

action or mechanical breakage cannot be excluded. systems and the characteristic ‘‘triad’’ pattern of the base

D and E), membrane

Among the decapods, the gonopore opercula of female of the three microtubular arms ( Fig. 4

Brachyura had been more extensively studied than in the nucleus is homogeneous and granular with visible fibrillar

D and E). The

Anomura. In particular, gonopores of female brachyurans chromatin.

close under several mechanisms ( Hartnoll 1968, 2006 ; Thompson and McLay 2005 ): (1) simple closure by muscle

3.5. Phylogenetic analysis action, like in Acanthocyclus Lucas, 1844, Jonas Hombron & Jacquinot, 1846, Bellia H. Milne Edwards, 1848,

The heuristic analysis of the 21 diogenid taxa using 19 Erimacrus Benedict, 1892 and Telmessus White, 1846 (see spermatozoal and spermatophore characters gave 43

Thompson and McLay 2005 ); (2) closure by soft oper- equally parsimonious trees after the reweighting proce-

culum, like in Heterozius A. Milne Edwards, 1867, Peltarion dure, rooted using the outgroup method. The cladogram

Hombron & Jacquinot, 1846, Pteropeltarion Dell, 1972, shown in Fig. 5 is the strict consensus of the 43 MP trees;

Trichopeltarion A. Milne Edwards, 1880, Pseudocorystes H. the bootstrap values are reported above the branches.

Milne Edwards, 1837 (see Thompson and McLay 2005 ), The pagurid hermit crab, P. bernhardus, is designated as

Uca vocans (see Nakasone et al. 1983 ; Salmon 1984 ) and the outgroup and the tree length=20 steps; consistency

Uca lactea (see Murai et al. 1987 ; Goshima and Murai index (CI)=0.75; retention index=0.91.

1988 ); or (3) closure by calcified operculum, like in Corystoides Lucas, 1844 and Corystes Bosc, 1802 (see Thompson and McLay 2005 ). When the operculum is calcified, it locally decalcifies at certain times permitting

4. Discussion

internal fertilization, as it happens in the families Dorippi- dae and Grapsidae (see Brockerhoff and McLay 2005a, b,

c ) and in Uca pugnax Smith, 1870 (see Greenspan 1982 ), Ilyoplax pusilla De Haan, 1835 (see Henmi and Murai In Paguristes eremita the male gonopores are not

4.1. Gonopores

1999 ), and Macrophthalmus hirtipes Heller, 1862 (see raised on any type of external prolongation of the coxae

Jennings et al. 2000 ).

(such as genital papillae) or of the ejaculatory ducts In contrast with other hermit crab species, in (such as elongate sexual tubes). Therefore it seems very

D. pugilator ( Manjo´n-Cabeza and Garcia likely that during copulation the males of P. eremita

particular

C. erythropus ( Tirelli et al. 2007 ), ejaculate spermatophores from the flush gonopores

Raso 2000 ) and

Isocheles sawayai Forest & de Saint Laurent, 1968 onto, or close to, the gonopores on the ventral face of

( Mantelatto et al. 2009 ), P. eremita essentially lacks any the coxae of pereopod 3 of the female, as reported by

setae immediately surrounding the male genital aperture Hess and Bauer (2002) for Clibanarius vittatus and by

of the gonopore and, instead, has little groups of setae Tirelli et al. (2007) for Clibanarius erythropus. So, as

located at a distance, as reported for Calcinus tibicen generally accepted for all hermit crabs (see Tudge and

( Amadio and Mantelatto, 2009 ). These setae may have Lemaitre 2004, 2006 ), fertilization is external.

a main function as active sensors of the female

T. Tirelli et al. / Zoologischer Anzeiger 248 (2010) 299–312

Fig. 5. Phylogenetic analysis: the strict consensus tree of 43 MP trees with Pagurus bernhardus as outgroup and rescaled consistency (RC) reweighted according to Farris’s (1969, 1988) iterative protocol. Tree length=20, CI=0.75, RI=0.91. Bootstrap values are above the branches.

cephalothorax and genital openings during spermato- for it is currently lacking. A sensorial function has also been phore transfer, an essential role during reproduction as

suggested for some of the setae found on the extensive underlined by Tirelli et al. (2007) for the ones

sexual tube of the Australian endemic hermit crab, surrounding the

Micropagurus acantholepis (Stimpson, 1858) (see Tudge sory roles have been previously inferred for the

C. erythropus gonopore. Similar sen-

and Lemaitre 2004 ). Although the presence of these setae is pleopodal (gonopodal) setae in some male brachyurans

evident, their sensorial function and real role during ( Beninger et al. 1991 ; Elner and Beninger 1992, 1995 ;

reproduction may only be proven through careful ablation Tsuchida and Fujikura 2000 ).

experiments.

Even though there is no insertion of any appendage The annulate setae without setules and annulate setae associated with the gonopores in hermit crabs, an important

with setules ( Watling 1989 ) present on the coxae of male sensory role for the male coxal setae is expected during

P. eremita show pronounced size differences, more so mating and spermatophore placement, but direct evidence

than distinct morphological ones. The shallow long-

T. Tirelli et al. / Zoologischer Anzeiger 248 (2010) 299–312

itudinal grooves that extend from their base to the Each one of the paired, lobe-like testes, is composed conspicuous annulus are unlikely to be preferential

of cystic structures, and continues into a tubular duct, flowing channels to convey spermatophores close to the

the vas deferens. Throughout its length the vas deferens female gonopore, as the setae do not immediately

has an inner secretory epithelium composed of cells surround the genital aperture. This is in contrast to a

containing cisternae of rough endoplasmic reticulum similarly suggested function of longitudinal grooves

and an outer muscular layer, in agreement with the present along the ejaculatory duct of some crab

description given by Kronenberger et al. (2004) for the gonopods ( Beninger et al. 1988, 1991 ; Tsuchida and

anomuran Galathea intermedia Liljeborg, 1851 and by Fujikura 2000 ; Moriyasu et al. 2002 ). The shallow setal

Manjo´n-Cabeza and Garcia Raso (2000) for the grooves may instead correspond to insertions by muscle

diogenid

D. pugilator.

groups, as suggested by Felgenhauer (1992) , because The vas deferens is made up of four zones, as seen in these setae probably need to move in all directions

other decapods such as the shrimp Pleoticus muelleri during mating. This muscle insertion hypothesis, as

(Bate, 1888) (see Diaz et al. 2002 ), the crayfish Cherax already reported for

Erichson, 1846 (see Talbot and Beach 1989 ) and the seems to be supported by the fact that grooves are

C. erythropus ( Tirelli et al. 2007 )

D. pugilator (see Manjo´n-Cabeza present only on the basal portion of the setae. However,

diogenid hermit crabs

and Garcia Raso 2000 ), Calcinus tubularis (see Tirelli this muscle insertion hypothesis still needs to be

et al. 2006b ), and Clibanarius erythropus (see Tirelli et al. confirmed by histological and TEM investigations. An

2007 ). As in these other described diogenids, there is a alternative hypothesis could be that the longitudinal

narrow collecting tubule producing the secretion that grooves may be artifacts of the progressive extrusion

envelops the spermatic mass, and in the same portion (lengthening) of setae through the cuticle during their

spermatozoa are carried towards the proximal zone, as growth ( Tirelli et al. 2007 ).

stated previously by Kooda-Cisco and Talbot (1986) , The medium and long, annulate, setae show various

Ro et al. (1990) , and Tirelli et al. (2006b, 2007) . The setules in their terminal portion that may serve as

spermatophore ampulla starts to be built in the mechanical functions during spermatophore transfer.

proximal zone, where the sperm mass becomes sub- While, in contrast with what is reported for

divided into successive portions, as already noted for ( Tirelli et al. 2007 ), they do not serve in connecting setae

C. erythropus

Enoplometopus A. Milne Edwards, 1862 sp. (see Haley to each other to form a setal tube, as there are too few of

1984 ), Homarus Weber, 1795 sp. (see Kooda-Cisco and them and they are not close enough to each other.

Talbot 1986 ), Galathea intermedia Liljeborg, 1851 (see These speculations about the possible functional

C. tubularis (see Tirelli et al. differences between the two types of setae ( sensu

Kronenberger et al. 2004 ),

C. erythropus (see Tirelli et al. 2007 ). In the Watling 1989 ) undoubtely need further investigation

2006b ) and

medial zone, the spermatophore is completed, showing at the histological and TEM level in order to be

the definitive ampulla shape, a moderately short stalk confirmed.

and a foot. In the distal zone the mature spermato- phores are simply stored, waiting to be transferred to the female during copulation ( Dudenhausen and Talbot 1983 ; Kooda-Cisco and Talbot 1986 ; Talbot and Beach

4.2. Testes, vasa deferentia and stages of 1989 ; Ro et al. 1990 ; Tirelli et al. 2006b, 2007 ). spermatophore maturation

Male specimens of P. eremita have their reproductive

4.3. Spermatophore

organs in the pleon. The internal reproductive apparatus is dorsal to the hepatopancreas and midgut. Its external

The present study supports the hypothesis that light morphology is similar to that of other hermit crabs, in

microscope observations of spermatophores can be used terms of components and position in the abdomen

successfully to distinguish hermit crab families of the ( McLaughlin 1980 ). They are long and highly coiled as

Paguroidea, and even within the family Diogenidae, in already reported for other hermit crabs ( Mouchet 1931 ;

agreement with Tudge (1991) and Tirelli and Pessani Matthews 1953, 1956 ; Greenwood 1972 ; Manjo´n-Cabeza

and Garcia Raso 2000 ; Scelzo et al. 2004 ; Tirelli et al. P. eremita exhibits spermatophores with the classic 2006b, 2007, 2008 ; Amadio and Mantelatto, 2009 ;

tripartite structure typical of anomurans and their Mantelatto et al. 2009 ).

general morphology described here is in agreement with As intact testes and vasa deferentia were dissected a

the description of the spermatophores of P. eremita greater number of coils, were observed, compared to

given by Mouchet (1931) . In immature spermatophores Mouchet (1931) , probably because the technique used

an accessory ampulla, close to the stalk, is present, as here preserved the spatial orientation of the entire

reported by Mouchet (1931) . The accessory ampulla is a reproductive tract ( Tirelli et al. 2006b, 2007 ).

typical element of the mature spermatophores of

T. Tirelli et al. / Zoologischer Anzeiger 248 (2010) 299–312

pagurids, parapagurids, and lithodids ( Tudge 1991, P. pollicaris=52 mm, P. bernhardus=50 mm, and 1999a ; Tudge et al. 1998a ), taxa in which the sperma-

P. excavatus=79 mm).

tophores do not undergo a significant stalk elongation. The ultrastructure of the ampulla wall of P. eremita is In agreement with Tudge (1999a) , the accessory

homogeneously granular, therefore generally in agree- ampullae are byproducts of the process of main

ment with those described for Pagurus hirtimanus White, spermatophore production within the vas deferens.

1847, P. prideaux, and Porcellanopagurus Filhol, 1885 During spermatophore formation, a continuous sperm

( Tudge 1999b ). Moreover, P. eremita ampulla walls column is periodically constricted by the vas deferens

show also a thin external fibrillar layer. musculature. The joining sperm column is retained

In conclusion, it seems possible to recognize sperma- throughout the remainder of spermatophore formation

tophores produced by P. eremita from the ones and sometimes only detaches from one of the adjacent

produced by other hermit crabs on the basis of their spermatophores. If stalk elongation follows ampulla

morphometry, in agreement with Scelzo et al. (2004) . It formation (as in the majority of diogenids), the sperm

seems also possible to recognize them from spermato- column material is stretched, and becomes progressively

phores produced by other members of the same family, thinner until the two ampullae of adjacent spermato-

as already shown by Tirelli and Pessani (2007) for the phores are completely separate. If stalk elongation is

C. tubularis, C. erythropus, and D. pugilator. absent, as in pagurids, parapagurids, and lithodids, the

diogenids

But it should be noted that Amadio and Mantelatto joining sperm column is incorporated into the short

(2009) found differences in spermatophore morphology stalk, appearing as the accessory ampullae.

due to hermit crab size and the different vas deferens In the present study the pedestal, although partially

regions considered.

covered by the protective structures which envelop the spermatophore, was observed, while Mouchet (1931) did not describe this portion of the spermatophore. The

4.4. Spermatozoa

absence of observation and description of the pedestal by Mouchet (1931) is probably due to the fact that

The spermatozoa of P. eremita show the typical Mouchet did not succeed in obtaining spermatophores

morphology of anomurans sperm: (1) an ovoid to completely free from the mucilage which envelopes

elongate acrosomal vesicle organized into concentric them, and simply reported a cord like structure

zones; (2) an electron-dense operculum capping the connecting the spermatophores one to the others, while

acrosomal vesicle; (3) a perforatorial chamber partially our observations place the cord like structure at the base

or wholly penetrating the acrosomal vesicle; (4) cyto- of the pedestal.

plasm with degenerate mitochondria and membrane The spermatophore of P. eremita superficially resem-

systems; (5) a generally diffuse posterior nucleus; and (6) bles the general morphology reported in some pagurids,

three or more microtubular arms emanating from the including Pagurus bernhardus (see Tudge, 1999a ),

cytoplasm (see Jamieson 1991 and Jamieson and Tudge Pagurus cuanensis Bell, 1845 (see Mouchet 1931 ),

2000 for reviews; Tudge et al. 2001 , Tirelli et al. 2006b, Pagurus excavatus (Herbst, 1791) (see Mouchet 1931 ;

Tudge 1999a ), Pagurus longicarpus Say, 1817 (see Tudge The typical acrosome vescicle shape recorded for the 1999a ), Pagurus pollicaris Say, 1817 (see Tudge 1999a ),

Anomura, varies from spherical to cylindrical, with a and Pagurus prideaux Leach, 1815 (see Mouchet 1931 ),

length:width ratio of approximately 1 or more, and has but the spermatophores of P. eremita, even if looking

been reported for all anomurans studied to date, except generally like the spermatophores of some pagurids

for the symmetrical hermit crab Pylocheles A. Milne when mature, lack the accessory ampulla. This latter

Edwards, 1880 ( Tudge et al. 2001 ). P. eremita, has a structure is present in immature spermatophores of

length:width ratio of 1.3 and a slightly ovoid acrosome P. eremita though.

shape, and is in total agreeement with data reported in Moreover, the total length of the spermatophore of

the literature ( Tudge 1995b, 1997 ; Scelzo et al. 2006 ). P. eremita is shorter than that of P. longicarpus and

Apart from these general anomuran characteristics, P. pollicaris, half the length of the spermatophores

the sperm cells of P. eremita show some similarities with of P. bernhardus, and 2/5’s of spermatophore length of

the spermatozoa described for the genus Clibanarius P. excavatus (P. eremita=144.3 mm, P. longicarpus=158

( Tudge 1992, 1995a, b ; Tudge and Justine 1994 ). In fact, m m, P. pollicaris=152 mm, P. bernhardus=250 mm and

P. eremita show a quite large and ovoid acrosome, P. excavatus=315 mm). As expected, the ampulla length

which constitutes almost the entire sperm volume, and a has an analogous trend ( P. eremita=92.6 mm, P. long-

perforatorial chamber, which resembles the one de- icarpus=97 mm, P. pollicaris=113 mm, P. bernhardus=220

scribed for Clibanarius. The spermatozoa of P. eremita m m, and P. excavatus=284 mm). The ampulla width though

are also smaller than Clibanarius species, being 3.6 mm is similar in all the above-mentioned species, except for

wide by 4.7 mm long, while the smallest Clibanarius P. excavatus (P. eremita=45.2 mm, P. longicarpus=43 mm,

spermatozoa are those produced by

C. erythropus, being

T. Tirelli et al. / Zoologischer Anzeiger 248 (2010) 299–312

P. eremita spermatozoa lack the dense from it. The setae seem to have two functions: assisting perforatorial ring at the base of the acrosomal vesicle

in conveying spermatophores to the female gonopores (found in Clibanarius spermatozoa), and do not show

and/or working as active sensors of the female any microvillar projections on the interior wall of the

cephalothorax and genital openings. perforatorial chamber. This last characteristic has been

In all four species, testes merge into a tubular duct described in all the other investigated diogenids, except a

made up of four zones, where spermatophores undergo member of the genus Cancellus H. Milne Edwards, 1836

different maturation phases. The spermatophore am- ( Tudge 1995a, b ) and Loxopagurus loxochelis ( Scelzo

pulla is constituted of two halves meeting at the lateral et al. 2006 ). Moreover, compared to all the other

ridge, which, together with the ampulla wall, shows a diogenids investigated, except for L. loxochelis ( Scelzo

C. tubularis ( Tirelli et al. et al. 2006 ), the spermatozoa of P. eremita have one

mostly fibrillar ultrastructure in

D. pugilator more exteriormost layer constituting the acrosome: the

2006b ),

C. erythropus ( Tudge 1999b ), and

( Tirelli et al. 2008 ) and a homogenously granular one in peripheral acrosome zone. This external zone has been

P. eremita (present paper). The ampulla shape varies in previously described, among the anomurans for the

the four species from round/ovoidal to quite long and hippids Emerita talpoida Say, 1817 and Hippa pacifica

narrow ( P. eremita); in C. erythropus lateral processes Dana, 1852 (see Tudge et al. 1999 ), and among the

are present, as in the other members of the same genus brachyurans for Cancer pagurus Linnaeus, 1758 (see

described up to now. The stalk is generally long and thin Tudge et al. 1994 ), Segonzacia mesatlantica (Williams,

C. tubularis) and the foot quite large. 1988) and Austinograea alayseae Guinot, 1990 (see

(except for

Spermatozoa have a large acrosomal vesicle, whose Tudge et al. 1998b ), for example. Furthermore,

shape varies among the four species, posteriorly P. eremita spermatozoa show, inside the perforatorial

penetrated by the perforatorial chamber ( Tudge and chamber, some structures resembling the perforatorial

Justine 1994 ; Tirelli et al. 2006b, 2007, 2008 ). The tubules already reported for some brachyurans like

perforatorial chamber shows microvillus-like projec-

C. pagurus ( Tudge et al. 1994 ), S. mesatlantica, and tions, extending radially into the lumen, except for

A. alayseae ( Tudge et al. 1998b ). P. eremita. The acrosome shows an inner and an outer In conclusion, the spermatophore morphology is most

D. pugilator similar to that reported for some pagurids, except for

acrosome zone in all four species. Only in

there is also a thick ray zone ( Tirelli et al. 2008 ). the fact that when mature P. eremita spermatophores

D. pugilator, the various acrosome zones, lack the accessory ampulla, but the spermatozoal

Moreover, in

in transverse section, have a circular profile at the ultrastructure does not support this pagurid relation-

perforatorial chamber level and a trilobed profile ship. P. eremita spermatozoa, instead, show a certain

immediately beneath the subopercular zone ( Tirelli number of characters typical of diogenids including the

C. tubularis, instead, there is an area apomorphy of the Clibanarius spermatozoon – the bulb

et al. 2008 ). In

that looks like the typical acrosome ray zone but the shape of the perforatorial chamber ( Tudge 1992, 1995a,

homology between this area and the acrosome ray zone

b , 1997; Tudge and Justine 1994 ). Also the absence of seems uncertain ( Tudge 1992, 1995a, b , 1997; Jamieson microvillar projections in the perforatorial chamber has

and Tudge 2000 , Tirelli et al. 2006b ). Only in P. eremita been similarly reported for the diogenid L. loxochelis

is a peripheral acrosome zone present. All four species ( Scelzo et al. 2006 ). Therefore, P. eremita spermatozoal

show an acrosome anteriorly delimited by a highly ultrastructure does not provide any clear apomorphies,

electron-dense operculum, beneath which there is a that separate it from the other diogenid genera already

subopercular zone; a cytoplasmic region enveloping the described but shares some characters with select

base of the acrosomal vesicle, with only a small portion diogenid genera.

connected with the perforatorial chamber; spherical As this research is the last of a series regarding the

mitochondria, cristate and degenerate membrane sys- description of the male reproductive apparatus of

tems and the characteristic ‘‘triad’’ pattern of the base of

the three microtubular arms; and finally a homogeneous Tirelli et al. 2007 ),

C. tubularis (see Tirelli et al. 2006b ),

C. erythropus (see

D. pugilator (see Tirelli et al. 2008 ),

and granular nucleus.

and P. eremita (present study), it is possible to underline the similarities and the differences among the reproduc- tive apparatus of these species.

The male gonopores of

C. erythropus ( Tirelli et al.

4.5. Phylogenetic analysis

D. pugilator ( Manjo´n-Cabeza and Garcia Raso 2000 ), and P. eremita are not raised on any type of

The addition of the herein studied species, P. eremita, external prolongation of the coxae or ejaculatory ducts

C. tibicen ( Amadio and and show a thin, possibly non- or weakly calcified

and the recently studied

Mantelatto, 2009 ), to the previous diogenid spermato- membrane closing it and numerous setae around it

zoal and spermatophore matrix ( Tirelli et al. 2008 ) made (

C. erythropus and D. pugilator) or at a short distance some difference to the phylogenetic tree of this group.

T. Tirelli et al. / Zoologischer Anzeiger 248 (2010) 299–312

The tree presented here ( Fig. 5 ) shows similarity (as Bauer, R.T., 1976. Mating behaviour and spermatophore expected) to the Bayesian consensus tree and Quartet

transfer in the shrimp Heptacarpus pictus (Stimpson) Puzzling parsimony tree (both in their figure 6) of Tirelli

(Decapoda: Caridea: Hippolytidae). J. Nat. Hist. 10, 415– et al. (2008) . P. eremita was simply inserted between the

monophyletic Clibanarius clade and remainder of the Bauer, R.T., 1986. Phylogenetic trends in sperm transfer and diogenid taxa. Fig. 5 shows (and Tirelli et al. 2008 ),

storage complexity in decapod crustaceans. J. Crust. Biol. though the low support values, a basal position of the

genus Clibanarius and its monophyly, and the weaker Bauer, R.T., 1991. Sperm transfer and storage structures in substructure of this clade probably reflects their

penaeoid shrimps. A functional and phylogenetic perspec- biogeography; Pacific versus Atlantic (see Tirelli et al.

tive. In: Bauer, R.T., Martin, J.W. (Eds.), Crustacean 2008 for comments).

Sexual Biology. Columbia University Press, New York, pp. Such a basal position of the genus Clibanarius is also

supported by the molecular analyses performed by Beninger, P.G., Elner, R.W., Poussart, Y., 1991. Gonopods of Morrison et al. (2002) and Mantelatto et al. (2006) . In

the majid crab Chionoecetes opilio (O. Fabricius) (Decapo- the current phylogenetic reconstruction, as previously da: Majidae) and a hypothesis for fertilization. J. Crust. mentioned, the position of Biol. 11, 217–228. P. eremita is uncertain between

Beninger, P.G., Elner, R.W., Foyle, T., Odense, P., 1988. Clibanarius and all the other diogenid genera. This fluidity Functional anatomy of the male reproductive system and of placement is also demonstrated in the molecular

the female spermatheca in the snow crab Chionoecetes opilio phylogeny of Mantelatto et al. (2006) , where their

(O. Fabricius) (Decapoda: Majidae) and a hypothesis for monophyletic Paguristes clade variously allied with the

fertilization. J. Crust. Biol. 8, 322–332. other diogenid taxa according to which cladistic metho-

Brockerhoff, A.M., McLay, C.L., 2005a. Mating behaviour, dology (NJ, MP or Bayesian) was applied. From a

female receptivity and male–male competition in the somatic morphological perspective the genera Clibanarius

intertidal crab Hemigrapsus sexdentatus (Brachyura: Grap- and Paguristes are quite similar to one another (e.g. equal

sidae). Mar. Ecol. Prog. Ser. 290, 179–191. or subequal chelae compared to the majority of the

Brockerhoff, A.M., McLay, C.L., 2005b. Comparative analy- diogenids which have noticeably larger left chelae) and so

sis of the mating strategies in grapsid crabs with special their adjacent placement in this tree of reproductive

references to the intertidal crabs Cyclograpsus lavauxi and characters is not so surprising. Inherent in this analysis,

Helice crassa (Decapoda: Grapsidae) from New Zealand. J. like many using solely reproductive characters, are the

Crust. Biol. 25, 507–520.

problems of low clade support and within-genus poly- Brockerhoff, A.M., McLay, C.L., 2005c. Factors influencing tomies (e.g. the genus Calcinus in Fig. 5 ) associated with

the onset and duration of receptivity of female purple rock small numbers of characters, limited taxonomic sampling,

crabs, Hemigrapsus sexdentatus (Brachyura: Grapsidae). J. and biased biogeographic sampling. Extending the

Exp. Mar. Biol. Ecol. 314, 123–135. analysis to more species within each genus, taxa from

Diaz, A.C., Fernandez Gimenez, A.V., Petriella, A.M., other geographic regions (this analysis is biased towards

Fenucci, J.L., 2002. Morphological and functional study the Indo-West Pacific by two-thirds), other morphological

of the male reproductive tract in the shrimp Pleoticus structures and characters, and even including molecular

muelleri Bate (Decapoda, Penaeoidea). Inv. Repr. Dev. 42, sequence data for these genera/species, would make an

interesting comparison for the phylogeny generated here Dudehausen, E.E., Talbot, P., 1983. An ultrastructural from reproductive characters alone.

comparison of soft and hardened spermatophores from the crayfish Pacifastacus leniusculus Dana. Can. J. Zool. 61, 182–194.

Elner, R.W., Beninger, P.G., 1992. The reproductive biology

Acknowledgements

of snow crab, Chionoecetes opilio: a synthesis of recent contributions. Am. Zool. 32, 524–533.

The authors would like to thank Professor Ezio Elner, R.W., Beninger, P.G., 1995. Multiple reproductive Campantico for assistance with transmission electron

strategies in snow crab, Chionoecetes opilio: physiological microscopy and the anonymous reviewers for improve-

pathways and behavioral plasticity. J. Exp. Mar. Biol. Ecol. ments to the manuscript.

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